Quantitative In Silico Evaluation of Allergenic Proteins from Anacardium occidentale,Carya illinoinensis,Juglans regia and Pistacia vera and Their Epitopes as Precursors of Bioactive Peptides

The aim of the study presented here was to determine if there is a correlation between the presence of specific protein domains within tree nut allergens or tree nut allergen epitopes and the frequency of bioactive fragments and the predicted susceptibility to enzymatic digestion in allergenic proteins from tree nuts of cashew (Anacardium occidentale), pecan (Carya illinoinensis), English walnut (Juglans regia) and pistachio (Pistacia vera) plants. These bioactive peptides are distributed along the length of the protein and are not enriched in IgE epitope sequences. Classification of proteins as bioactive peptide precursors based on the presence of specific protein domains may be a promising approach. Proteins possessing a vicilin, N-terminal family domain, or napin domain contain a relatively low occurrence of bioactive fragments. In contrast, proteins possessing the cupin 1 domain without the vicilin N-terminal family domain contain a relatively high total frequency of bioactive fragments and predicted release of bioactive fragments by the joint action of pepsin, trypsin, and chymotrypsin. This approach could be utilized in food science to simplify the selection of protein domains enriched for bioactive peptides.     

Evolution of functional diversity in the cupin superfamily

The cupin superfamily of proteins is among the most functionally diverse of any described to date. It was named on the basis of the conserved β-barrel fold (‘cupa’ is the Latin term for a small barrel), and comprises both enzymatic and non-enzymatic members, which have either one or two cupin domains. Within the conserved tertiary structure, the variety of biochemical function is provided by minor variation of the residues in the active site and the identity of the bound metal ion. This review discusses the advantages of this particular scaffold and provides an evolutionary analysis of 18 different subclasses within the cupin superfamily.     

Food allergen detection by mass spectrometry: From common to novel protein ingredients

Food allergens are molecules, mainly proteins, that trigger immune responses in susceptible individuals upon consumption even when they would otherwise be harmless. Symptoms of a food allergy can range from mild to acute; this last effect is a severe and potentially life-threatening reaction. The European Union (EU) has identified 14 common food allergens, but new allergens are likely to emerge with constantly changing food habits. Mass spectrometry (MS) is a promising alternative to traditional antibody-based assays for quantifying multiple allergenic proteins in complex matrices with high sensitivity and selectivity. Here, the main allergenic proteins and the advantages and drawbacks of some MS acquisition protocols, such as multiple reaction monitoring (MRM) and data-dependent analysis (DDA) for identifying and quantifying common allergenic proteins in processed foodstuffs are summarized. Sections dedicated to novel foods like microalgae and insects as new sources of allergenic proteins are included, emphasizing the significance of establishing stable marker peptides and validated methods using database searches. The discussion involves the in-silico digestion of allergenic proteins, providing insights into their potential impact on immunogenicity. Finally, case studies focussing on microalgae highlight the value of MS as an effective analytical tool for ensuring regulatory compliance throughout the food control chain.     

Solution structure of a methionine-rich 2S albumin from sunflower seeds: relationship to its allergenic and emulsifying properties

The three-dimensional structure in aqueous solution of SFA-8, a 2S albumin 103-residue protein from seeds of sunflower (Helianthus anuus L.), has been determined by NMR methods. An almost complete (1)H resonance assignment was accomplished from analysis of two-dimensional (2D) COSY and 2D TOCSY spectra, and the structure was computed by using restrained molecular dynamics on the basis of 1393 upper limit distance constraints derived from NOE cross-correlation intensities measured in 2D NOESY spectra. In contrast with most other 2S albumins, SFA-8 consists of a single polypeptide chain without any cleavage in the segment of residues 30-46. The computed structures exhibited an rmsd radius of 0.52 A for the backbone structural core (residues 11-30 and 46-101) and 1.01 A for the side chain heavy atoms. The resulting structure consists of five amphipathic helices arranged in a right-handed superhelix, a folding motif first observed in nonspecific lipid transfer (nsLTP) proteins, and common to other 2S albumins. In contrast to nsLTP proteins, neither SFA-8 nor RicC3 (a 2S albumin from castor bean) has an internal cavity that is able to host a lipid molecule, which results from an exchange in the pairing of disulfide bridges in the CXC segment. Both 2S albumins and nonspecific lipid transfer proteins belong to the prolamin superfamily, which includes a number of important food allergens. Differences in the extension and solvent exposition of the so-called "hypervariable loop" (which connects helices III and IV) in SFA-8 and RicC3 may be responsible for the different allergenic properties of the two proteins. SFA-8 has been shown to form highly stable emulsions with oil/water mixtures. We propose that these properties may be determined partly by a hydrophobic patch at the surface of the protein which consists of five methionines that partially hide the Trp76 residue. The flexibility of the loop which contains Trp76 and the hydrophobicity of the whole environment may favor a conformational change, by which the Trp76 side chain may become inserted into the oil phase.     

Thiol dioxygenases: unique families of cupin proteins

Proteins in the cupin superfamily have a wide range of biological functions in archaea, bacteria and eukaryotes. Although proteins in the cupin superfamily show very low overall sequence similarity, they all contain two short but partially conserved cupin sequence motifs separated by a less conserved intermotif region that varies both in length and amino acid sequence. Furthermore, these proteins all share a common architecture described as a six-stranded β-barrel core, and this canonical cupin or “jelly roll” β-barrel is formed with cupin motif 1, the intermotif region, and cupin motif 2 each forming two of the core six β-strands in the folded protein structure. The recently obtained crystal structures of cysteine dioxygenase (CDO), with contains conserved cupin motifs, show that it has the predicted canonical cupin β-barrel fold. Although there had been no reports of CDO activity in prokaryotes, we identified a number of bacterial cupin proteins of unknown function that share low similarity with mammalian CDO and that conserve many residues in the active-site pocket of CDO. Putative bacterial CDOs predicted to have CDO activity were shown to have similar substrate specificity and kinetic parameters as eukaryotic CDOs. Information gleaned from crystal structures of mammalian CDO along with sequence information for homologs shown to have CDO activity facilitated the identification of a CDO family fingerprint motif. One key feature of the CDO fingerprint motif is that the canonical metal-binding glutamate residue in cupin motif 1 is replaced by a cysteine (in mammalian CDOs) or by a glycine (bacterial CDOs). The recent report that some putative bacterial CDO homologs are actually 3-mercaptopropionate dioxygenases suggests that the CDO family may include proteins with specificities for other thiol substrates. A paralog of CDO in mammals was also identified and shown to be the other mammalian thiol dioxygenase, cysteamine dioxygenase (ADO). A tentative fingerprint motif for ADOs, or DUF1637 family members, is proposed. In ADOs, the conserved glutamate residue in cupin motif 1 is replaced by either glycine or valine. Both ADOs and CDOs appear to represent unique clades within the cupin superfamily.     

Specific conformational epitope features of pathogenesis-related proteins mediating cross-reactivity between pollen and food allergens

Selected members of plant pathogenesis-related and seed storage proteins represent specific groups of proteins with potential characteristics of allergens. Efforts to understand the mechanism by which pathogenesis-related proteins mediate a broad cross-reactivity in pollen-plant food allergens are still limited. In this study, computational biology approach was used to reveal specific structural implications and conservation of different epitopes from members of Bet v 1 and nsLTP protein families mediating cross-reactivity between pollen and food (fruits, vegetables, legumes, and nut/seeds) allergens. A commonly shared epitope conservation was found among all pollen and food Bet v 1 and nsLTP protein families, respectively. However, other allergenic epitopes were also specifically detected in each family. The implication of these conserved epitopes in a broad cross-reactivity for allergy clinical trials is here discussed.     

Germin and Germin-like Proteins: Evolution,Structure, and Function

Germin and germin-like proteins (GLPs) are encoded by a family of genes found in all plants. They are part of the cupin superfamily of biochemically diverse proteins, a superfamily that has a conserved tertiary structure, though with limited similarity in primary sequence. The subgroups of GLPs have different enzyme functions that include the two hydrogen peroxide–generating enzymes, oxalate oxidase (OxO) and superoxide dismutase. This review summarizes the sequence and structural details of GLPs and also discusses their evolutionary progression, particularly their amplification in gene number during the evolution of the land plants. In terms of function, the GLPs are known to be differentially expressed during specific periods of plant growth and development, a pattern of evolutionary subfunctionalization. They are also implicated in the response of plants to biotic (viruses, bacteria, mycorrhizae, fungi, insects, nematodes, and parasitic plants) and abiotic (salt, heat/cold, drought, nutrient, and metal) stress. Most detailed data come from studies of fungal pathogenesis in cereals. This involvement with the protection of plants from environmental stress of various types has led to numerous plant breeding studies that have found links between GLPs and QTLs for disease and stress resistance. In addition the OxO enzyme has considerable commercial significance, based principally on its use in the medical diagnosis of oxalate concentration in plasma and urine. Finally, this review provides information on the nutritional importance of these proteins in the human diet, as several members are known to be allergenic, a feature related to their thermal stability and evolutionary connection to the seed storage proteins, also members of the cupin superfamily.     

Pyr c 1, the major allergen from pear (Pyrus communis), is a new member of the Bet v 1 allergen family

Pear is known as an allergenic food involved in the ‘oral allergy syndrome’ which affects a high percentage of patients allergic to birch pollen. The aim of this study was to clone the major allergen of this fruit, to express it as bacterial recombinant protein and to study its allergenic properties in relation to homologous proteins and natural allergen extracts. The coding region of the cDNA was obtained by a PCR strategy, cloned, and the allergen was expressed as His-Tag fusion protein. The fusion peptide was removed by treatment with cyanogen bromide. Purified non-fusion protein was subjected to allergenicity testing by the enzyme allergosorbent test (EAST), Western blotting, competitive inhibition assays, and basophil histamine release. The deduced protein sequence shared a high degree of identity with other major allergens from fruits, nuts, vegetables, and pollen, and with a family of PR-10 pathogenesis related proteins. The recombinant (r) protein was recognised by specific IgE from sera of all pear-allergic patients (n=16) investigated in this study. Hence, the allergen was classified as a major allergen and named Pyr c 1. The IgE binding characteristics of rPyr c 1 appeared to be similar to the natural pear protein, as was demonstrated by EAST-inhibition and Western blot-inhibition experiments. Moreover, the biological activity of rPyr c 1 was equal to that of pear extract, as indicated by basophil histamine release in two patients allergic to pears. The related major allergens Bet v 1 from birch pollen and Mal d 1 from apple inhibited to a high degree the binding of IgE to Pyr c 1, whereas Api g 1 from celery, also belonging to this family, had little inhibitory effects, indicating epitope differences between Bet v 1-related food allergens. Unlimited amounts of pure rPyr c 1 are now available for studies on the structure and epitopes of pollen-related food allergens. Moreover, the allergen may serve as stable and standardised diagnostic material.     

Structure and stability of 2S albumin-type peanut allergens: implications for the severity of peanut allergic reactions

Resistance to proteolytic enzymes and heat is thought to be a prerequisite property of food allergens. Allergens from peanut (Arachis hypogaea) are the most frequent cause of fatal food allergic reactions. The allergenic 2S albumin Ara h 2 and the homologous minor allergen Ara h 6 were studied at the molecular level with regard to allergenic potency of native and protease-treated allergen. A high-resolution solution structure of the protease-resistant core of Ara h 6 was determined by NMR spectroscopy, and homology modelling was applied to generate an Ara h 2 structure. Ara h 2 appeared to be the more potent allergen, even though the two peanut allergens share substantial cross-reactivity. Both allergens contain cores that are highly resistant to proteolytic digestion and to temperatures of up to 100 degrees C. Even though IgE antibody-binding capacity was reduced by protease treatment, the mediator release from a functional equivalent of a mast cell or basophil, the humanized RBL (rat basophilic leukaemia) cell, demonstrated that this reduction in IgE antibody-binding capacity does not necessarily translate into reduced allergenic potency. Native Ara h 2 and Ara h 6 have virtually identical allergenic potency as compared with the allergens that were treated with digestive enzymes. The folds of the allergenic cores are virtually identical with each other and with the fold of the corresponding regions in the undigested proteins. The extreme immunological stability of the core structures of Ara h 2 and Ara h 6 provides an explanation for the persistence of the allergenic potency even after food processing.     

Genetic modification and plant food allergens: risks and benefits

Plant genetic engineering has the potential to both introduce new allergenic proteins into foods and remove established allergens. A number of allergenic plant proteins have been characterized, showing that many are related to proteins which have potentially valuable properties for use in nutritional enhancement, food processing and crop protection. It is therefore important to monitor the allergenic potential of proteins used for plant genetic engineering and major biotechnology companies have established systems for this. Current technology allows gene expression to be down-regulated using antisense or co-suppression and future developments may allow targeted gene mutation or gene replacement. However, the application of this technology may be limited at least in the short term by the presence of multiple allergens and their contribution to food processing or other properties. Furthermore, the long-term stability of these systems needs to be established as reversion could have serious consequences.     

Solution structure and stability against digestion of rproBnIb, a recombinant 2S albumin from rapeseed: relationship to its allergenic properties

NMR spectroscopy has been used to determine the solution structure of the precursor form of the recombinant napin BnIb, rproBnIb, a 2S albumin, 109-residue protein from the seeds of Brassica napus. More than 90% of the side-chain proton resonances were unambiguously assigned from the analysis of two-dimensional correlation (COSY), total correlation (TOCSY), and nuclear Overhauser effect (NOESY) spectra. The final structures were computed by using restrained molecular dynamics on the basis of 1316 upper-limit distance constraints derived from NOE cross-correlation intensities. The computed structures exhibited a root-mean-square deviation (RMSD) radius of 0.66 A for the backbone and 1.16 A for the side-chain heavy atoms of the structural core. The resulting structure consists of five amphipathic helices arranged in a right-handed super helix, a folding motif found in other proteins of the prolamin superfamily. As in the case of the mature protein, the recombinant precursor behaves as a plant food allergen. To trace out the origin and characteristics of its allergenic properties, rproBnIb was assayed against simulated gastric fluid and found to be very resistant to proteolysis. Also, heat treatment of the protein followed up to 85 degrees C by circular dichroism showed a very limited unfolding, which was recovered after cooling to 20 degrees C, indicating a high thermal stability. These results suggest that rproBnIb, as other 2S albumins, may be able to reach the gut immune system intact. A comparison of the putative epitopes against IgE antibodies of the three members of the prolamine family [2S albumins, nonspecific lipid transfer proteins (nsLTPs), and alpha-amylase/trypsin inhibitors] indicates that there are not common surfaces of interaction with IgE. Though the epitopes appear to be located in different regions of the proteins, they do comply with the requirements of being solvent-exposed and flexible.     

Immunological identification and characterization of individual food allergens

Food allergies of type-I-allergy are immunoglobulin E (IgE) mediated and caused by certain proteins or glycoproteins, which are called food allergens. An analytical marker of allergens is the IgE-reactivity to these substances. Normally food allergens are minor components in allergenic source material, which consist of a huge number of chemical different substances. Thus allergen extraction, separation and immunological detection methods are described which identify and characterize individual food allergens by a minimum of manipulation. Favoured separation methods of allergenic extracts are electrophoretic ones allowing the combination of highly resolved protein separations with immunological detection methods subsumed by the term immunoblotting. These techniques are a useful basis to characterize allergens by chemical methods. Once the primary protein structure of a food allergen is established, the way is cleared for the identification of epitopes. Epitopes are immunological detectable parts of a protein or glycoprotein generating the interface between chemical structure and immune-system. The nature of epitopes may differ, for instance, can be conformational, continuous, or built up by glycoconjugates, which determine the stability of food allergens, especially in the case of food processing. Progress in identification and characterization of food allergens will improve diagnostics and therapy of food allergy.     

苦荞全基因组11S种子储藏蛋白基因的鉴定及表达分析

以苦荞（Fagopyrum tataricum（L.）Gaertn）全基因组数据为平台,采用生物信息学方法,挖掘出9个11S种子储藏蛋白基因,并对其定位、蛋白结构、系统发育及表达模式进行了分析。结果表明,苦荞9个11S种子储藏蛋白基因编码的蛋白长度为189~914 aa,等电点位于5.18~9.82之间,分子量为21.27~103.33 kD;定位分析结果显示,这些成员位于苦荞基因组的6条连锁群上（Megascaffold2/5以及scaffold77/344/395/861）;序列比对分析发现,除了1个11S种子储藏蛋白sample1_00009513-RA具有1个cupin保守结构域外,其余8个都含有2个cupin结构域,并且在cupin保守结构域中,苦荞和拟南芥（Arabidopsis thaliana（L.）Heynh）共有14个保守的氨基酸残基;蛋白结构预测表明,苦荞11S种子储藏蛋白的结构具有2种类型;苦荞与其它6个物种[拟南芥、花生（Arachis hypogaea Linn.）、大豆（Glycine max（Linn.）Merr.）、杏仁（Armeniaca vulgaris Lam.）、胡桃（Juglans regia L.）和芝麻（Sesamum indicum Linn.）]11S种子储藏蛋白以及苦荞过敏蛋白（TBb和TBt）系统发育分析结果表明,这些蛋白可以分为3类,共具有4对旁系同源蛋白和3对直系同源蛋白;与已报道的苦荞过敏性储藏蛋白以及其它5个物种（花生、大豆、杏仁、胡桃和芝麻）的11S过敏蛋白比较发现,5个11S种子储藏蛋白（sample1_00013128-RA、sample1_00013130-RA、sample1_00021677-RA、sample1_00021668-RA和sample1_00021674-RA）与苦荞2个过敏蛋白的同源性较高,同时它们与胡桃11S过敏蛋白的同源性最高,但尚需进一步实验来确定这5个成员是否为食物过敏原;RNA-Seq转录组数据显示,4个基因（sample1_00018411-RA、sample1_00026786-RA、sample1_00021674-RA、sample1_00022718-RA）在2种荞麦属植物的灌浆期种子中表达水平较高,且在‘大苦1号’中的表达水平要高于‘大甜1号’。     

Identification of the 7S and 11S globulins as percutaneously sensitizing soybean allergens as demonstrated through epidermal application of crude soybean extract

Cutaneous exposure to food allergens can predispose individuals to food allergies. Soybean, a major allergenic food, is an ingredient in various cosmetic products. However, the types of soybean proteins that are percutaneously sensitizing in humans or animal models remain unknown. In this study, BALB/c mice were dorsally shaved and epicutaneously exposed to a crude soybean extract including sodium dodecyl sulfate or distilled water alone. Specific IgEs secreted in response to 7S globulin (Gly m 5), 11S globulin (Gly m 6), Gly m 3, and Gly m 4 were measured using enzyme-linked immunosorbent assays or immunoblots. Exposure to soybean extract elicited the secretion of soybean-specific IgEs. Of the soybean proteins, 7S and 11S globulins acted as percutaneous sensitizers in 6/9 mice (67%). Additionally, IgE bound specifically and preferentially to the 7S globulin β subunit. In conclusion, this is the first report to identify percutaneously sensitizing soybean allergens in a mouse model.     

High-yield expression in Escherichia coli, purification, and characterization of properly folded major peanut allergen Ara h 2

Allergic reactions to peanuts are a serious health problem because of their high prevalence, associated with potential severity, and chronicity. One of the three major allergens in peanut, Ara h 2, is a member of the conglutin family of seed storage proteins. Ara h 2 shows high sequence homology to proteins of the 2S albumin family. Presently, only very few structural data from allergenic proteins of this family exist. For a detailed understanding of the molecular mechanisms of food-induced allergies and for the development of therapeutic strategies knowledge of the high-resolution three-dimensional structure of allergenic proteins is essential. We report a method for the efficient large-scale preparation of properly folded Ara h 2 for structural studies and report CD-spectroscopic data. In contrast to other allergenic 2S albumins, Ara h 2 exists as a single continuous polypeptide chain in peanut seeds, and thus heterologous expression in Escherichia coli was possible. Ara h 2 was expressed as Trx-His-tag fusion protein in E. coli Origami (DE3), a modified E. coli strain with oxidizing cytoplasm which allows the formation of disulfide bridges. It could be shown that recombinant Ara h 2, thus overexpressed and purified, and the allergen isolated from peanuts are identical as judged from immunoblotting, analytical HPLC, and circular dichroism spectra.     

Homology modeling of allergenic cyclophilins: IgE-binding site and structural basis of cross-reactivity

Cross-reactivity among allergens is of considerable scientific as well as clinical interest. Proteins belonging to the allergenic cyclophilin family share a high degree of sequence homology and are cross-reactive. Until date no three-dimensional structural information is available on these proteins and the shared structural features of the epitopes which are the most important determinants of cross-reactivity. Cyclophilins are also known to bind with the immuno-suppressive drug cyclosporin. Comparative molecular modeling of these allergenic cyclophilin proteins of different sources was performed in order to investigate the structural basis of their cross-reactivity. All the proteins studied revealed a similarity in the shape of the cross-reactive epitopes with varying degrees of accessibility. Cyclosporin binding and allergenic properties of these proteins were also found to be structurally related.     

Computational analysis of the relationship between allergenicity and digestibility of allergenic proteins in simulated gastric fluid

Background  

Safety assessment of genetically modified (GM) food, with regard to allergenic potential of transgene-encoded xenoproteins, typically involves several different methods, evaluation by digestibility being one thereof. However, there are still debates about whether the allergenicity of food allergens is related to their resistance to digestion by the gastric fluid. The disagreements may in part stem from classification of allergens only by their sources, which we believe is inadequate, and the difficulties in achieving identical experimental conditions for studying digestion by simulated gastric fluid (SGF) so that results can be compared. Here, we reclassify allergenic food allergens into alimentary canal-sensitized (ACS) and non-alimentary canal-sensitized (NACS) allergens and use a computational model that simulates gastric fluid digestion to analyze the digestibilities of these two types.     

Search for the determinants of allergenicity in proteins of the lipocalin family

Three different lines of analysis have been applied to approach the problem of the allergenicity of certain proteins: biological functions, molecular structures and immunological properties. It is immediately obvious that these three are interdependent. The lipocalin family of proteins includes a significant number of allergens. A considerable amount of data is already available of lipocalins and some insights about allergenic determinants can now be presented. However, more information on the molecular structures and immunological parameters of lipocalin allergens is required.